The present invention relates generally to polyphenolic proteins. More specifically it is concerned with a new and improved process for purifying and stabilizing catechol-containing proteins and with the stabilized proteinaceous materials obtained thereby.
As mentioned in my publication, The Journal of Biological Chemistry, Vol. 258, No. 5, pp. 2911-2915 (Mar. 10, 1983), several species of common marine mussels of the genus Mytilus secure themselves to solid substrates through a complex array of plaque-tipped collagenous byssal threads. The ends of these threads are rich in a polyphenolic adhesive substance that is mixed by the animal's foot with a curing enzyme (phenoloxidase) and a mucosubstance to provide a complex three-component natural adhesive system. The polyphenolic protein component of that system has been identified as a polymeric protein rich in 3,4-dihydroxyphenylalanine (dopa) (11 percent) and hydroxyproline (hyp) (13 percent). The amino acid composition of the polyphenolic proteins is reported by Waite and Tanzer in "Polyphenolic Substance of Mytilus edulis: Novel Adhesive Containing L-DOPA and Hydroxyproline" Science, Vol. 212, pp. 1038-1040 (1981). The disclosures within these publications are incorporated herein by reference.
These polyphenolic proteins are unique in their ability to adhere to substrates under the environmentally adverse and turbulent conditions in which the mussels exist. This is significant since, typically, adhesives are adversely affected by the presence of water on the substrates being adhered. Water competes with the adhesive for the surface, tends to hydrolyze the adhesive, and frequently plasticizes the adhesive. Accordingly, it is usually required that the substrate surfaces being adhered to be substantially free from water or other aqueous impurities. As can be appreciated, such conditions are not always possible, particularly for bioadhesives used in medical and dental applications and employing a wide variety of substrates such as those encountered when gluing or restoring fractured hard tissue in the body such as bone, cartilage, teeth, ligaments, blood vessels and the like.
The polyphenolic proteinaceous bioadhesive is also unusual in its superior strength characteristics which appear to be comparable to those achieved by synthetic cyanoacrylates. Since it can be applied to wet surfaces without prior drying, it may be considered to be superior to such adhesives. Further, the polyphenolic protein cures extremely rapidly, is nontoxic, and can be used in very fine or thin films exhibiting a coefficient of expansion similar to biological tissue.
As reported in the publications mentioned hereinbefore, the polyphenolic protein consists of a rather large polypeptide chain having a molecular weight of about 110,000 to 140,000 in which seven amino acids account for about 80 percent of all the amino acid residues within the peptide. A particular decapeptide sequence is given and it is stated that the reported sequence and related sequences may be repeated as often as 75 times in the polypeptide proteins. The reported presence of dopa and hydroxyprolines is unusual since dopa is only rarely encountered as a component of naturally occurring proteins and the hyroxyprolines are primarily associated with collagens having a high glycine content.
The isolation of the polyphenolic proteins reported hereinbefore involves treatment of dissected phenol glands of numerous mussels with a neutral salt buffer followed by extraction of the protein with acetic acid. As reported, the extraction is effective in providing a reasonable amount of the polyphenolic proteins. However, the acid soluble material at this stage of isolation and purification has a limited shelf life. This is believed to be due to many factors including the presence of collagen and dopa's susceptability to facile oxidation to its quinone moiety. Dopa is an o-diphenol and readily forms quinones and semiquinones by photolysis, autoxidation and enzyme catalysis. Additionally, the protein is very sensitive to the presence of transition metal elements and tends to irreversibly coalesce with other proteinaceous materials still present within the acetic acid extract. It is known that other o-diphenols can chelate various metals, such as copper, iron, manganese, zinc, and nickel with high affinity. This characteristic is believed to contribute to sclerotization of the o-diphenol proteins.
While ion exchange techniques have been attempted as a means of achieving greater purification, it is recognized that yields of the proteins deteriorate drastically as a result of the extensive adsorption of the proteins by the ion exchange medium. In fact, up to 70 percent of the applied polyphenolic proteins are not recovered when using this technique. Gel filtration of the proteins using a variety of chromatographic materials and buffers generally results in very low or negligible yields. Although some materials permit recovery of the protein, they typically provide a limited fractionation range and generally are not preferred for purifying the bioadhesive proteins.